Anti-prokineticin receptor (PROKR) antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to prokineticin receptors (PROKRs) and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human PROKR1 and/or PROKR2. The present invention includes antibodies that bind cell surface-expressed PROKR1 and/or PROKR2. In certain embodiments, the antibodies of the present invention are capable of blocking prokineticin (PK)-mediated activation of one or more PROKR. The antibodies of the invention are useful for the treatment of various diseases and disorders mediated by prokineticin signaling.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/078,024 filed Nov. 12, 2013, entitled “Anti-Prokineticin Receptor(PROKR) Antibodies and Uses Thereof” which claims the benefit under 35U.S.C. § 119(e) of U.S. provisional application Ser. Nos. 61/725,704,filed on Nov. 13, 2012 and 61/825,112, filed on May 20, 2013, thedisclosures of which are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which bind to and/or block a prokineticin receptor(PROKR1 and/or PROKR2), and methods of use thereof.

BACKGROUND

Prokineticins (PK1 and PK2) are secreted, multifunctional chemokine-likepeptides. Prokineticins exert their biological functions through theinteraction with two G-protein coupled receptors (GPCRs) referred to asprokineticin receptor 1 (PROKR1 or PKR1) and prokineticin receptor 2(PROKR2 or PKR2). PROKR1 and PROKR2 share approximately 87% overallamino acid sequence identity with one another. PK1 and PK2 each interactwith PROKR1 and PROKR2. At the cellular level, activation ofprokineticin receptors leads to calcium mobilization, stimulation ofphosphoinositide turnover and activation of the MAP kinase signalingpathway. At the multicellular level, prokineticins exhibit angiogenicactivity and induce cell proliferation and migration. Prokineticins andtheir receptors are associated with the development of several humancancers and have also been shown to participate in nociception and thetransmission of pain. (See, e.g., Monnier and Samson, 2010, CancerLetters 296:144-149; and Negri et al., 2009, Int. Rev. Neurobiol.85:145-157).

The prokineticin signaling system represents a potential target for thetreatment and/or prevention of a variety of diseases and disorders.Accordingly, a need exists in the art for novel therapeutic agents whichtarget and modulate one or more components of the prokineticin signalingpathway, such as the prokineticin receptors (PROKR1 and PROKR2).

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind prokineticinreceptors (PROKRs). The antibodies of the invention are useful, interalia, for inhibiting PROKR-mediated signal transduction and for treatingdiseases and disorders caused by or related to PROKR activity orprokineticin signaling. According to certain embodiments, the anti-PROKRantibodies of the present invention may be used to treat or attenuatepain in a subject in need thereof.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

The present invention provides anti-PROKR antibodies or antigen-bindingfragments thereof comprising a heavy chain variable region (HCVR) havingan amino acid sequence selected from the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 82, 98, 114, 130, 146, and 162, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a light chain variable region (LCVR)having an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, and 170, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, and 162/170.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, and 168, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR3 (LCDR3) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144,160, and 176, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,88/96, 104/112, 120/128, 136/144, 152/160, and 168/176.

The present invention also provides an antibody or fragment thereoffurther comprising a heavy chain CDR1 (HCDR1) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 20,36, 52, 68, 84, 100, 116, 132, 148, and 164, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 6,22, 38, 54, 70, 86, 102, 118, 134, 150, and 166, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; a light chain CDR1 (LCDR1) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, and 172, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR2 (LCDR2) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142,158, and 174, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g. H1M6386N);20-22-24-28-30-32 (e.g. H 2M6385N); 36-38-40-44-46-48 (e.g. H4H6663P);52-54-56-60-62-64 (e.g. H4H6669P); 68-70-72-76-78-80 (e.g. H4H6671P);84-86-88-92-94-96 (e.g. H4H6680P); 100-102-104-108-110-112 (e.g.H4H6690P); 116-118-120-124-126-128 (e.g. H4H6696P);132-134-136-140-142-144 (e.g. H4H6698P); 148-150-152-156-158-160 (e.g.,H4H6701P); and 164-166-168-172-174-176 (e.g. H4 H6706P).

In a related embodiment, the invention includes an anti-PROKR antibodyor antigen-binding fragment thereof, wherein the antibody or fragmentcomprises the heavy and light chain CDR domains derived from heavy andlight chain variable region (HCVR/LCVR) sequences selected from thegroup consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, 114/122, 130/138, 146/154, and 162/170. Methods and techniquesfor identifying CDRs within HCVR and LCVR amino acid sequences are wellknown in the art and can be used to identify CDRs within the specifiedHCVR and/or LCVR amino acid sequences disclosed herein. Exemplaryconventions that can be used to identify the boundaries of CDRs include,e.g., the Kabat definition, the Chothia definition, and the AbMdefinition. In general terms, the Kabat definition is based on sequencevariability, the Chothia definition is based on the location of thestructural loop regions, and the AbM definition is a compromise betweenthe Kabat and Chothia approaches. See, e.g., Kabat, “Sequences ofProteins of Immunological Interest,” National Institutes of Health,Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948(1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272(1989). Public databases are also available for identifying CDRsequences within an antibody. Once the CDRs of a reference antibody areidentified, new antibodies exhibiting identical or substantially similarbinding properties as the reference antibody can be easily made usingroutine methods in the art.

In another aspect, the invention provides nucleic acid moleculesencoding anti-PROKR antibodies or antigen-binding fragments thereof.Recombinant expression vectors carrying the nucleic acids of theinvention, and host cells into which such vectors have been introduced,are also encompassed by the invention, as are methods of producing theantibodies by culturing the host cells under conditions permittingproduction of the antibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, and 161, or a substantially identical sequence having at least90%, at least 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofcomprising a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137,153, and 169, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,23, 39, 55, 71, 87, 103, 119, 135, 151, and 167, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof; and a LCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 15,31, 47, 63, 79, 95, 111, 127, 143, 159, and 175, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof.

The present invention also provides an antibody or fragment thereofwhich further comprises a HCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83,99, 115, 131, 147, and 163, or a substantially identical sequence havingat least 90%, at least 95%, at least 98%, or at least 99% homologythereof; a HCDR2 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133,149, and 165, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof; a LCDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, and171, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and a LCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, and 173, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

According to certain embodiments, the antibody or fragment thereofcomprises the heavy and light chain CDR sequences encoded by the nucleicacid sequences of SEQ ID NOs: 1 and 9 (e.g. H1M6386N), 17 and 25 (e.g.H2M6385N), 33 and 41 (e.g. H4H6663P), 49 and 57 (e.g. H4H6669P), 65 and73 (e.g. H4H6671P), 81 and 89 (e.g. H4H6680P), 97 and 105 (e.g.H4H6690P), 113 and 121 (e.g. H4H6696P), 129 and 137 (e.g. H4H6698P), 145and 153 (e.g. H4H6701P), or 161 and 169 (e.g. H4H6706P).

The present invention includes anti-PROKR antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain.

In another aspect, the invention provides a pharmaceutical compositioncomprising an anti-PROKR antibody or antigen-binding fragment thereofand a pharmaceutically acceptable carrier. In a related aspect, theinvention features a composition comprising a combination of ananti-PROKR antibody and a second therapeutic agent. In one embodiment,the second therapeutic agent is any agent that is advantageouslycombined with an anti-PROKR antibody. Exemplary agents that may beadvantageously combined with an anti-PROKR antibody include, withoutlimitation, other agents that inhibit PROKR activity (including otherantibodies or antigen-binding fragments thereof, peptide inhibitors,small molecule antagonists, natural products, etc.) and/or agents whichdo not directly bind PROKR but nonetheless interfere with, block orattenuate PROKR-mediated activity or signal transduction.

In yet another aspect, the invention provides methods for inhibitingPROKR activity using an anti-PROKR antibody or antigen-binding portionof an antibody of the invention, wherein the therapeutic methodscomprise administering a therapeutically effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment of an antibody of the invention. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by removal, inhibition or reduction of PROKR activity. Theanti-PROKR antibodies or antibody fragments of the invention mayfunction to block the interaction of a PROKR with one or moreprokineticin.

The present invention also includes the use of an anti-PROKR antibody orantigen binding portion of an antibody of the invention in themanufacture of a medicament for the treatment of a disease or disorderrelated to or caused by PROKR activity in a patient.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1A depicts the extent of immobility time in seconds; FIG. 1Bdepicts the total distance traveled in cm; FIG. 1C depicts the rearingcounts; and FIG. 1D depicts the rearing time in seconds. All parameterswere measured over a 60 minute test period. The study summarizes openfield behaviors of mice subjected to a DSS-induced colitis model. Micewere treated with either: water alone, DSS alone, DSS+isotype controlantibody, or DSS+antibody H4H6385N, as indicated.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expressions “prokineticin receptor,” “PROKR,” “PKR,” and the like,as used herein, are intended to encompass both PROKR1 and PROKR2. Theterms “PROKR1” and “PROKR2” refer to the human PROKR1 or PROKR 2proteins unless specified as being from a non-human species (e.g.,“mouse PROKR1,” “mouse PROKR2,” “monkey PROKR1,” “monkey PROKR2,” etc.).Human PROKR1 has the amino acid sequence of SEQ ID NO:177. Human PROKR2has the amino acid sequence of SEQ ID NO:178. Mouse (Mus musculus)PROKR1 has the amino acid sequence as set forth in NCBI referencesequence number NP_067356.2; mouse PROKR2 has the amino acid sequence asset forth in NCBI reference sequence number NP_659193.3; rat (Rattusnorvegicus) PROKR1 has the amino acid sequence as set forth in NCBIreference sequence number NP_620433.1; rat PROKR2 has the amino acidsequence as set forth in NCBI reference sequence number NP_620434.1;cynomolgus monkey (Macaca fascicularis) PROKR1 has the amino acidsequence as set forth in GenBank accession number EHH55625.1; andcynomolgus monkey PROKR2 has the amino acid sequence as set forth inGenBank accession number EHH65528.1.

An “anti-PROKR antibody” means an antibody that specifically bindseither PROKR1 or PROKR2, or both PROKR1 and PROKR2. As used herein, “anantibody that binds PROKR” or an “anti-PROKR antibody” includesantibodies, and antigen-binding fragments thereof, that bind a solublefragment of PROKR protein (e.g., a polypeptide comprising the N-terminalextracellular portion of PROKR1 or PROKR2 [see, e.g., Example 4 herein],or one or more extracellular loops thereof). The expressions “anantibody that binds PROKR” or an “anti-PROKR antibody” also includeantibodies that bind cell surface-expressed PROKR1 and/or PROKR2. Theexpression “cell surface-expressed PROKR” means one or more PROKRprotein(s) that is/are expressed on the surface of a cell in vitro or invivo, such that at least a portion of the PROKR protein (e.g., theN-terminal extracellular portion and/or one or more extracellular loops)is/are exposed to the extracellular side of the cell membrane andaccessible to an antigen-binding portion of an antibody. “Cellsurface-expressed PROKRs” include PROKRs that are naturally expressed onthe surface of a cell as well as PROKRs that are artificially engineeredto be expressed on the surface of a cell.

An antibody that “specifically binds cell surface-expressed PROKR1”means an antibody that detectably binds cells that express PROKR1 on thecell surface but does not detectably bind equivalent cells that do notexpress PROKR1 on the cell surface. Likewise, an antibody that“specifically binds cell surface-expressed PROKR2” means an antibodythat detectably binds cells that express PROKR2 on the cell surface butdoes not detectably bind equivalent cells that do not express PROKR2 onthe cell surface. An exemplary method for assessing whether an antibodybinds cell surface-expressed PROKR1 and/or PROKR2 is flow cytometry(FACS), as illustrated in Example 3 herein.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., a PROKR). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-PROKR antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H) , V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) VL_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to a PROKR: (i) inhibits theinteraction between a prokineticin (e.g., PK1 or PK2) and the PROKR;(ii) inhibits or attenuates prokineticin-mediated activation of thePROKR; and/or (iii) results in inhibition of at least one biologicalfunction of the PROKR. The inhibition caused by a PROKR-neutralizing orblocking antibody need not be complete so long as it is detectable usingan appropriate assay. Exemplary assays for detecting PROKR inhibitionare known in the art and are illustrated in the working Examples herein.

The anti-PROKR antibodies disclosed herein may comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogennicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-PROKR antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-PROKR antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24:307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256:1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Biological Characteristics of the Antibodies

The present invention includes anti-PROKR antibodies and antigen-bindingfragments thereof that specifically bind cell surface-expressed PROKR1and/or cell surface-expressed PROKR2. For example, the present inventionprovides anti-PROKR antibodies that: (a) specifically bind cellsurface-expressed PROKR1 but not cell surface-expressed PROKR2; (b)specifically bind cell surface-expressed PROKR2 but not cellsurface-expressed PROKR1; or (c) specifically bind cellsurface-expressed PROKR1 and cell surface-expressed PROKR2. Anti-PROKRantibodies can be tested and evaluated for the ability to specificallybind a cell surface-expressed PROKR using any assay format that allowsfor the detection of antibody binding to cells that express a PROKR. Anexemplary assay format that can be used to determine whether an antibodyspecifically binds a cell surface-expressed PROKR is illustrated inExample 3 herein. In this Example, cells that normally do not expressPROKRs (e.g., HEK293 cells) are engineered to express PROKR1 or PROKR2,and antibody binding to the PROKR-expressing cells is determined by flowcytometry with detectably labeled secondary antibodies. “Specificantibody binding” to a cell surface-expressed PROKR means that thepercentage of cells that exhibit detectable binding by flow cytometry isgreater than 1%. An antibody that exhibits a binding percentage ofbetween 1% and 10% in this assay format is generally regarded as having“weak” binding, but is nonetheless considered an antibody that“specifically binds a cell surface-expressed PROKR” for purposes of thepresent disclosure. According to certain embodiments, however, specificantibody binding to a cell surface-expressed PROKR means that thepercentage of cells that exhibit detectable binding by flow cytometry isgreater than 10%, greater than 20%, greater than 30%, greater than 40%,greater than 50%, or more.

The present invention also includes anti-PROKR antibodies that bind oneor more soluble fragments of PROKR1 and/or PROKR2. For example,antibodies are provided herein which specifically bind a solublefragment of PROKR1 or PROKR2 comprising all or part of the N-terminalextracellular portion of the PROKR protein. Exemplary soluble PROKR1 andPROKR2 constructs of this type are illustrated in Example 4 herein. Asshown in Example 4, fusion proteins comprising amino acids 1-62 ofPROKR1 (SEQ ID NO:177) or amino acids 1-53 or PROKR2 (SEQ ID NO:178),fused to a human Fc component, were tested for binding to anti-PROKRantibodies by surface plasmon resonance (at 25° C. and pH 7.4). Using anassay format of Example 4, or a similar assay, the binding of anti-PROKRantibodies to soluble PROKR molecules can be quantified, e.g., in termsof equilibrium dissociation constant (K_(D)) and/or dissociationhalf-life (t½).

Thus, the present invention provides anti-PROKR antibodies that bindsoluble human PROKR1 (e.g., N-terminal portion) with a K_(D) of lessthan about 5 nM, less than about 3 nM, less than about 2 nM, less thanabout 1.5 nM, less than about 600 pM, less than about 550 pM, less thanabout 500 pM, less than about 450 pM, less than about 400 pM, less thanabout 350 pM, less than about 300 pM, less than about 250 pM, less thanabout 200 pM, less than about 150 pM, or less than about 100 pM asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 4 herein or a substantially similar assay.

The present invention also includes anti-PROKR antibodies andantigen-binding fragments thereof that bind soluble human PROKR1 (e.g.,N-terminal portion) with a dissociation half-life (t½) of greater thanabout 5 minutes, greater than about 10 minutes, greater than about 15minutes, greater than about 20 minutes, greater than about 25 minutes,greater than about 30 minutes, greater than about 35 minutes, greaterthan about 40 minutes, greater than about 45 minutes, greater than about50 minutes, greater than about 55 minutes, greater than about 60minutes, greater than about 75 minutes, greater than about 100 minutes,greater than about 150 minutes, greater than about 200 minutes, orgreater than about 250 minutes, or more, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 4 hereinor a substantially similar assay.

The present invention also provides anti-PROKR antibodies that bindsoluble human PROKR2 (e.g., N-terminal portion) with a K_(D) of lessthan about 150 nM, less than about 130 nM, less than about 100 nM, lessthan about 90 nM, less than about 80 nM, less than about 75 nM, lessthan about 70 nM, less than about 65 nM, less than about 60 nM, lessthan about 55 nM, less than about 50 nM, less than about 45 nM, lessthan about 40 nM, less than about 35 nM, less than about 30 nM, lessthan about 25 nM, less than about 20 nM, less than about 15 nM, lessthan about 10 nM, less than about 5 nM, less than about 3 nM, or less,as measured by surface plasmon resonance, e.g., using the assay formatas defined in Example 4 herein or a substantially similar assay.

The present invention also includes anti-PROKR antibodies andantigen-binding fragments thereof that bind soluble human PROKR2 (e.g.,N-terminal portion) with a dissociation half-life (t½) of greater thanabout 1 minute, greater than about 2 minutes, greater than about 3minutes, greater than about 4 minutes, greater than about 5 minutes,greater than about 10 minutes, greater than about 20 minutes, greaterthan about 30 minutes, greater than about 40 minutes, or more, asmeasured by surface plasmon resonance, e.g., using the assay format asdefined in Example 4 herein or a substantially similar assay.

The present invention also includes anti-PROKR antibodies andantigen-binding fragments thereof that block prokineticin-mediatedactivation of PROKR1 and/or PROKR2. The ability of anti-PROKR antibodiesto block prokineticin-mediated activation of PROKR1 and/or PROKR2 can bemeasured, e.g., using the assay format illustrated in Example 5 herein.In this assay, cells that do not normally express human PROKRs (i.e.,HEK293 cells) are engineered to express PROKR1 or PROKR2. In this assayformat, the extent of PROKR activation is indicated by calciummobilization following treatment with prokineticin-1 (PK1) orprokineticin-2 (PK2) (e.g. using a concentration of about 1 to 20 nM orPK1 or PK2), in the presence or absence of an anti-PROKR antibody.Inhibition of prokineticin-mediated PROKR activation in this assayformat is calculated as an IC₅₀ value (i.e., the concentration ofantibody needed to inhibit PK-mediated calcium flux by 50%) or as ablocking percentage. The present invention includes anti-PROKRantibodies that block: (a) PK1-mediated activation of PROKR1; (b)PK2-mediated activation of PROKR1; (c) PK1-mediated activation ofPROKR2; and/or (d) PK2-mediated activation of PROKR2. For example, thepresent invention includes anti-PROKR antibodies that block PK1-mediatedactivation of PROKR1 with an IC₅₀ of less than about 20 nM, less thanabout 18 nM, less than about 16 nM, less than about 14 nM, less thanabout 12 nM, less than about 10 nM, less than about 9 nM, less thanabout 8 nM, less than about 7 nM, or less than about 6 nM, or less, asmeasured using the assay format of Example 5 (e.g., using about 1 nM toabout 20 nM of PK1), or a substantially similar assay. The presentinvention also includes anti-PROKR antibodies that block PK2-mediatedactivation of PROKR1 with an IC₅₀ of less than about 60 nM, less thanabout 50 nM, less than about 20 nM, or less than about 20 nM, asmeasured using the assay format of Example 5 (e.g., using about 1 nM toabout 20 nM of PK2), or a substantially similar assay.

The present invention also includes anti-PROKR antibodies that inhibitor reduce pain response(s) in various animal pain models.

Other biological activities of the anti-PROKR antibodies of the presentinvention will be apparent to persons of ordinary skill in the art inlight of the working Examples set forth herein.

Epitope Mapping and Related Technologies

The present invention includes anti-PROKR antibodies which interact withone or more amino acids located within one or more regions or segmentsof the PROKR1 molecule selected from the group consisting of: (a) theN-terminal extracellular region (amino acids 1 to 62 of SEQ ID NO:177);(b) extracellular loop 1 (amino acids 120 to 146 of SEQ ID NO: 177); (c)extracellular loop 2 (amino acids 201 to 232 of SEQ ID NO:177); and/orextracellular loop 3 (amino acids 304 to 322 of SEQ ID NO:177).

The present invention also includes anti-PROKR antibodies which interactwith one or more amino acids located within one or more regions orsegments of the PROKR2 molecule selected from the group consisting of:(a) the N-terminal extracellular region (amino acids 1 to 54 of SEQ IDNO:178); (b) extracellular loop 1 (amino acids 110 to 137 of SEQ IDNO:178); (c) extracellular loop 2 (amino acids 193 to 221 of SEQ IDNO:178); and/or extracellular loop 3 (amino acids 297 to 310 of SEQ IDNO:178).

The epitope to which the antibodies bind may consist of a singlecontiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within anyof the aforementioned regions or segments of PROKR1 and/or PROKR2.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) located within one or more of theaforementioned regions or segments of a PROKR molecule. For example, theantibodies of the present invention may interact with one or more aminoacids located within the N-terminal extracellular region of PROKR1 aswell as one or more amino acids located within one or more extracellularloops of PROKR1.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

The present invention further includes anti-PROKR antibodies that bindto the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. H1M6386N, H2M6385N, H4H6663P, H4H6669P, H4H6671P,H4H6680P, H4H6690P, H4H6696P, H4H6698P, H4H6701P, H4H6706P, etc.).Likewise, the present invention also includes anti-PROKR antibodies thatcompete for binding to PROKR1 and/or PROKR2 with any of the specificexemplary antibodies described herein (e.g. H1M6386N, H2M6385N,H4H6663P, H4H6669P, H4H6671P, H4H6680P, H4H6690P, H4H6696P, H4H6698P,H4H6701P, H4H6706P, etc.).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-PROKR antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-PROKRantibody of the invention, the reference antibody is allowed to bind toa PROKR protein. Next, the ability of a test antibody to bind to thePROKR molecule is assessed. If the test antibody is able to bind to thePROKR following saturation binding with the reference anti-PROKRantibody, it can be concluded that the test antibody binds to adifferent epitope than the reference anti-PROKR antibody. On the otherhand, if the test antibody is not able to bind to the PROKR moleculefollowing saturation binding with the reference anti-PROKR antibody,then the test antibody may bind to the same epitope as the epitope boundby the reference anti-PROKR antibody of the invention. Additionalroutine experimentation (e.g., peptide mutation and binding analyses)can then be carried out to confirm whether the observed lack of bindingof the test antibody is in fact due to binding to the same epitope asthe reference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, Biacore, flow cytometry or any otherquantitative or qualitative antibody-binding assay available in the art.In accordance with certain embodiments of the present invention, twoantibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-,10-, 20-or 100-fold excess of one antibody inhibits binding of the otherby at least 50% but preferably 75%, 90% or even 99% as measured in acompetitive binding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antibodies are deemed to bind tothe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

To determine if an antibody competes for binding with a referenceanti-PROKR antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to a PROKR protein under saturatingconditions followed by assessment of binding of the test antibody to thePROKR molecule. In a second orientation, the test antibody is allowed tobind to a PROKR molecule under saturating conditions followed byassessment of binding of the reference antibody to the PROKR molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the PROKR molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to thePROKR. As will be appreciated by a person of ordinary skill in the art,an antibody that competes for binding with a reference antibody may notnecessarily bind to the same epitope as the reference antibody, but maysterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to a human PROKR.

Using VELOCIMMUNE™ technology or other similar methods for generatingmonoclonal antibodies, high affinity chimeric antibodies to PROKR areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-PROKR antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bind ahuman PROKR. Such variant antibodies and antibody fragments comprise oneor more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-PROKR antibody-encoding DNA sequences of the present inventionencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-PROKR antibody or antibody fragment that isessentially bioequivalent to an anti-PROKR antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-PROKR antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-PROKR antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention includes anti-PROKR antibodies that bind to ahuman PROKR (e.g., cell surface-expressed human PROKR1 and/or cellsurface expressed human PROKR2) but not to PROKRs from other species.The present invention also includes anti-PROKR antibodies that bind to ahuman PROKR (e.g., cell surface-expressed human PROKR1 and/or cellsurface expressed human PROKR2) and also bind to one or more PROKRproteins from one or more non-human species. The present invention alsoincludes anti-PROKR antibodies that block prokineticin-mediatedactivation of human PROKR1 and/or human PROKR2 but do not blockprokineticin-mediated activation of one or more non-human PROKRs. Thepresent invention also includes anti-PROKR antibodies that blockprokineticin-mediated activation of human PROKR1 and/or human PROKR2 andalso block prokineticin-mediated activation of one or more non-humanPROKRs.

For example, the anti-PROKR antibodies of the invention may bind toand/or block human PROKR1 and/or human PROKR2, and may bind and/or block(or not bind or not block as the case may be) one or more of mouse, rat,guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow,horse, camel, cynomologous, marmoset, rhesus or chimpanzee PROKR1 orPROKR2. For example, as shown in Example 5 herein, certain exemplaryantibodies of the present invention block PK1-mediated activation ofhuman PROKR1 as well as PK1-mediated activation of monkey PROKR1 (e.g.,H4H6696, H4H6698, H4H6701 and H4H6385). On the other hand, antibodyH1M6386 exhibited potent blocking of PK1-mediated activation of humanPROKR1 but did not exhibit any detectable blocking of PK1-mediatedactivation of monkey PROKR1. Other cross-reactivity/cross-blockingpatterns of the exemplary anti-PROKR antibodies of the present inventionwill be apparent to a person of ordinary skill in the art upon review ofthe working examples provided herein.

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-PROKR antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor a human PROKR or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety. The present invention includesbispecific antibodies comprising a first antigen-binding domain thatspecifically binds PROKR1 and a second antigen-binding domain thatspecifically binds PROKR2.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E,L358M, N384S, K392N, V397M, and V4221 by EU) in the case of IgG1antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221by EU) inthe case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, andV821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 byEU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Therapeutic Formulation and Administration

The present invention provides pharmaceutical compositions comprisingthe anti-PROKR antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with PROKR activity in an adult patient, it may beadvantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering anti-PROKRantibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25 ™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-PROKR antibody. The therapeutic composition can comprise any of theanti-PROKR antibodies, or fragments thereof, as disclosed herein. Asused herein, the expression “a subject in need thereof” means a human ornon-human animal that exhibits one or more symptoms or indicia of adisease or disorder associated with or caused by PROKR activity, or whootherwise would benefit from an inhibition or reduction PROKR signaling.

Exemplary diseases and disorders that can be treated with the anti-PROKRantibodies of the present invention include pain conditions (e.g.,acute, chronic, or breakthrough pain). Exemplary types of painconditions that are treatable with the anti-PROKR antibodies of thepresent invention include nociceptive pain, visceral pain (e.g., painfrom inflammatory bowel disease/irritable bowel syndrome, interstitialcystitis, pancreatitis, endometriosis, chronic pelvic pain syndrome,etc.), as well as pain associated with inflammation (e.g., inflammatorymuscle pain), post-operative incision (e.g., post-surgical pain),neuropathy (e.g., diabetic neuropathy), sciatica, post-herpeticneuralgia, myofascial pain syndromes, arthritis, sickle cell, entericnerve ischemia, claudication pain, bone fracture, burn, osteoporoticfracture, gout, migraine headache, fibromyalgia, complex regional painsyndrome, acute herpetic pain, etc.

The anti-PROKR antibodies of the present invention are also useful fortreating or preventing cancer-associated pain. “Cancer-associated pain”includes, e.g., bone cancer pain, including pain from cancer that hasmetastasized to bone (e.g., breast cancer, prostate cancer, lung cancer,sarcoma, kidney cancer, multiple myeloma, etc.). “Cancer-associatedpain” also includes pain more generally associated with cancerousconditions such as, e.g., renal cell carcinoma, pancreatic carcinoma,breast cancer, head and neck cancer, prostate cancer, malignant gliomas,osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma,multiple myeloma, ovarian cancer, small cell lung cancer, non-small celllung cancer, synovial sarcoma, thyroid cancer, or melanoma.

The antibodies of the present invention may also be useful in treatingdiseases and disorders associated with and/or caused by pathologicalangiogenesis (e.g., tumors, angiogenic eye disorders, etc.). Otherdiseases and disorders that may be treated using the anti-PROKRantibodies of the present invention include, e.g., disorders of thegastrointestinal tract (e.g., involving smooth muscle contraction),Hirschsprung disease, polycystic ovarian syndrome, Kallman syndrome,rheumatoid arthritis, and osteoarthritis. The anti-PROKR antibodies ofthe present invention may also be used for fertility applications.

Combination Therapies and Formulations

The present invention provides methods which comprise administering apharmaceutical composition comprising any of the exemplary anti-PROKRantibodies described herein in combination with one or more additionaltherapeutic agents. Exemplary additional therapeutic agents that may becombined with or administered in combination with an anti-PROKR antibodyof the present invention include, e.g., other pain-attenuating biologicssuch as anti-NGF antibodies, anti-PAR2 antibodies, anti-ASIC antibodies(e.g., anti-ASIC1, anti-ASIC2, anti-ASIC3, and anti-ASIC4 antibodies),anti-GFRα antibodies, as well as non-biologic therapeutic agents such asantivirals, antibiotics, analgesics, corticosteroids, opioids, and/orNSAIDs.

The anti-PROKR antibodies of the present invention may also be combinedwith or administered in combination with one or more cancer therapeuticagent(s) such as, e.g., an EGFR antagonist (e.g., an anti-EGFR antibody[e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR[e.g., gefitinib or erlotinib]), an antagonist of another EGFR familymember such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 orErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody thatspecifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMETantibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-rafinhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-αinhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g.,an anti-PDGFRI-β antibody), a VEGF antagonist (e.g., a VEGF-Trap, see,e.g., U.S. Pat. No. 7,087,411 (also referred to herein as a“VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), a FOLH1 antagonist (e.g., an anti-FOLH1antibody), a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 orSTEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti-STEAP2antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLNantagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., ananti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakinantibody), a CD20 antagonist (e.g., an anti-CD20 antibody such asrituximab), etc.

Other agents that may be beneficially administered in combination withthe anti-PROKR antibodies of the invention include cytokine inhibitors,including small-molecule cytokine inhibitors and antibodies that bind tocytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11,IL-12, IL-13, IL-17, IL-18, or antagonists of their respectivereceptors, as well as anti-IgE antibodies, anti-TNF antibodies, etc.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan anti-PROKR antibody of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-PROKR antibody “in combination with” anadditional therapeutically active component). The present inventionincludes pharmaceutical compositions in which an anti-PROKR antibody ofthe present invention is co-formulated with one or more of theadditional therapeutically active component(s) as described elsewhereherein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-PROKR antibody may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of an anti-PROKR antibody of the invention. As used herein,“sequentially administering” means that each dose of anti-PROKR antibodyis administered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of ananti-PROKR antibody, followed by one or more secondary doses of theanti-PROKR antibody, and optionally followed by one or more tertiarydoses of the anti-PROKR antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-PROKR antibody ofthe invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-PROKR antibody, but generally may differ from one another in termsof frequency of administration. In certain embodiments, however, theamount of anti-PROKR antibody contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½21,21½, 22, 22½, 23, 23½, 24, 24½25, 25½26, 26½, or more) weeks after theimmediately preceding dose. The phrase “the immediately preceding dose,”as used herein, means, in a sequence of multiple administrations, thedose of anti-PROKR antibody which is administered to a patient prior tothe administration of the very next dose in the sequence with nointervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PROKR antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-PROKR antibodies of the present invention may also be used todetect and/or measure one or more PROKR protein(s), or PROKR-expressingcells in a sample, e.g., for diagnostic purposes. For example, ananti-PROKR antibody, or fragment thereof, may be used to diagnose acondition or disease characterized by aberrant expression (e.g.,over-expression, under-expression, lack of expression, etc.) of PROKR1or PROKR2. Exemplary diagnostic assays for PROKR may comprise, e.g.,contacting a sample, obtained from a patient, with an anti-PROKRantibody of the invention, wherein the anti-PROKR antibody is labeledwith a detectable label or reporter molecule. Alternatively, anunlabeled anti-PROKR antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure PROKR in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in PROKR diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of PROKR protein, orfragments thereof, under normal or pathological conditions. Generally,levels of PROKR in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal PROKR levels or activity) will be measured to initiallyestablish a baseline, or standard, level of PROKR. This baseline levelof PROKR can then be compared against the levels of PROKR measured insamples obtained from individuals suspected of having a PROKR relateddisease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Prokineticin Receptors

To generate anti-PROKR antibodies, a VELOCIMMUNE® mouse, comprising DNAencoding human Immunoglobulin heavy and kappa light chain variableregions, was immunized with a mouse fibroblast cell line (MG87)engineered to express either human PROKR1 or human PROKR2. The antibodyimmune response was monitored by a cell binding assay using cellsengineered to express human PROKRs. When a desired immune response wasachieved, splenocytes were harvested and fused with mouse myeloma cellsto preserve their viability and form hybridoma cell lines. The hybridomacell lines were screened and selected to identify cell lines thatproduce PROKR-specific antibodies. Using this technique anti-PROKRchimeric antibodies (i.e., antibodies possessing human variable domainsand mouse constant domains) were obtained, including antibodiesdesignated H1M6386N and H2M6385N.

Anti-PROKR antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in US 2007/0280945A1. Using this method, several fully human anti-PROKR antibodies (i.e.,antibodies possessing human variable domains and human constant domains)were obtained; exemplary antibodies generated in this manner weredesignated as follows: H4H6663P, H4H6669P, H4H6671P, H4H6680P, H4H6690P,H4H6696P, H4H6698P, H4H6701P, and H4H6706P.

Certain biological properties of the exemplary anti-PROKR antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-PROKR antibodies and their correspondingantibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 6386N 2 4 6 8 10 12 14 16 6385N 18 20 22 24 26 28 3032 6663P 34 36 38 40 42 44 46 48 6669P 50 52 54 56 58 60 62 64 6671P 6668 70 72 74 76 78 80 6680P 82 84 86 88 90 92 94 96 6690P 98 100 102 104106 108 110 112 6696P 114 116 118 120 122 124 126 128 6698P 130 132 134136 138 140 142 144 6701P 146 148 150 152 154 156 158 160 6706P 162 164166 168 170 172 174 176

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” “H4H”), followed by a numericalidentifier (e.g. “6386,” “6385,” “6663,” etc. as shown in Table 1),followed by a “P” or “N” suffix. Thus, according to this nomenclature,an antibody may be referred to herein as, e.g., “H1M6386N,” “H2M6385N,”“H4H6663P,” etc. The Fc prefixes on the antibody designations usedherein indicate the particular Fc region of the antibody. For example,an “H1M” antibody has a mouse IgG1 Fc, whereas an “H4H” antibody has ahuman IgG4 Fc. As will be appreciated by a person of ordinary skill inthe art, an antibody with a particular IgG isotype (e.g., “H4H”) can beconverted to an antibody with a different IgG isotype (e.g., H1H, H1M,H2M, etc.) using routine methods; but in any event, the variable domains(including the CDRs)—which are indicated by the numerical identifiersshown in Table 1—will remain the same, and the binding properties areexpected to be identical or substantially similar regardless of thenature of the Fc domain.

Example 3 Determination of the Binding of Anti-PROKR Antibodies toCell-Surface PROKR1 and PROKR2 by Flow Cytometry

Anti-PROKR antibodies generated in accordance with Example 1 were testedfor the ability to bind to human, mouse, and monkey PROKR1 and PROKR2.For these experiments, HEK293 cells were engineered to express: humanPROKR1; human PROKR2; mouse PROKR1; mouse PROKR2; cynomolgus monkeyPROKR1; or cynomolgus monkey PROKR2. Binding of anti-human PROKRantibodies to the PROKR-expressing cell lines was measured by flowcytometry. The experimental protocol is set forth below, and the resultsare summarized in Table 2.

Adherent cells were collected using 1 mM EDTA in PBS, then washed, andre-suspended in cold PBS containing 5% FBS. For the binding experiments,each anti-PROKR antibody was added to 250,000 cells in 500 μl of PBSwith 5% FBS (final antibody concentration of 13 nM). After incubationfor 20 minutes at room temperature, the cells were washed with PBScontaining 5% FBS. A secondary antibody, recognizing either human(Jackson Immuno Research, #115-135-205) or mouse Fc (BD Pharmigen,#550826) and conjugated to allophycyanin, was then added to the cellmixture at a final concentration of 13.3 nM. After incubating for 20minutes on ice, the cells were washed and resuspended in PBS containing5% FBS and then sorted and analyzed on a FACSCalibur (BD Biosciences)flow cytometer to determine relative binding by the candidateantibodies. The cell samples containing secondary antibody alone wereused as negative control for FACS gating. Histograms of cells stainedwith anti-human PROKR antibodies were compared with cells stained withsecondary alone. The percentage of cells exhibiting a PROKR FACS bindingsignal greater than the signal observed with secondary antibody alone(“percentage binding”) was calculated by FlowJo software (Tree Star).The samples stained with anti-PROKR antibodies were recorded as FACSpositive (“Pos” in Table 1) when percentage binding was greater than10%. The samples stained with anti-human PROKR antibodies were recordedas FACS negative (“Neg” in Table 1) when percentage binding was lowerthan 1% or when signal was detected on parental cells (i.e., backgroundsignal). The samples stained with anti-human PROKR antibody wererecorded as weak (“Weak” in Table 1) when percentage binding was between1%-10%. ND=not determined.

TABLE 2 Binding of Anti-PROKR Antibodies to HEK293 Cells ExpressingHuman, Mouse or Monkey PROKR1 or PROKR2 Measured by Flow Cytometry HumanHuman Mouse Mouse Monkey Monkey Antibody PROKR1 PROKR2 PROKR1 PROKR2PROKR1 PROKR2 H4H6663P Pos Pos ND ND ND ND H4H6669P Neg Neg ND ND ND NDH4H6671P Neg Neg ND ND ND ND H4H6680P Neg Neg ND ND ND ND H4H6690P PosNeg ND ND ND ND H4H6696P Pos Neg ND ND ND ND H4H6698P Pos Pos Pos PosPos Pos H4H6701P Pos Pos Pos Pos Pos Pos H4H6706P Neg Neg ND ND ND NDH2M6385N Weak Pos Neg Pos Neg Pos H1M6386N Weak Pos Neg Neg Neg Neg

As shown in Table 2, five antibodies bound to cells expressing humanPROKR1 and to cells expressing human PROKR2: H4H6663P, H4H6698P,H4H6701P, H2M6385N and H1M6386N (with H2M6385N and H1M6386N binding onlyweakly to cells expressing human PROKR1). Two of the antibodies testedbound to cells expressing human PROKR1 but not to cells expressing humanPROKR2: H4H6690P and H4H6696P.

Antibodies H4H6698P and H4H6701P, which bound to cells expressing humanPROKR1 and cell expressing human PROKR2, also exhibited positive bindingto cells expressing mouse and monkey PROKR1 and PROKR2. AntibodyH2M6385N, which bound only weakly to cells expressing human PROKR1, wasnegative for binding to mouse and monkey PROKR1 but was positive forbinding to mouse and monkey PROKR2.

Thus, as illustrated by this example, the present invention includes:(a) antibodies that specifically bind human PROKR1 and human PROKR2; (b)antibodies that specifically bind human PROKR1 and human PROKR2, as wellas PROKR1 and PROKR2 from non-human species (e.g., mouse and monkey);and (c) antibodies that specifically bind human PROKR1 but not humanPROKR2. Anti-PROKR antibodies with binding specificity patterns, otherthan those specifically illustrated in this Example, are alsocontemplated within the scope of the present invention.

Example 4 Determination of the Equilibrium Binding Constants forAnti-PROKR Antibodies Binding to PROKR1 and PROKR2 by Surface PlasmonResonance (Biacore)

Equilibrium dissociation constants (K_(D) values) were determined forantigen binding to selected purified anti-PROKR antibodies generated inaccordance with Example 1 by surface kinetics using a real-time surfaceplasmon resonance biosensor (Biacore 4000) assay at 25° C. For theseexperiments, antibody was captured on either a goat anti-mouse IgGpolyclonal antibody (GE Healthcare, #BR-1008-38) or a mouse anti-humanIgG (Fc) monoclonal antibody (GE Healthcare, #BR-1008-39) surfacecreated through direct amine coupling to a Biacore CM5 sensor chip.Kinetic experiments were carried out using HBS-EP (10 mM HEPES, 150 mMNaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) as both the runningbuffer and the sample buffer. Antigen-antibody association rates weremeasured by injecting 2 concentrations (25 and 100 nM) of both humanPROKR1(1-62)-hFc (SEQ ID NO:179) and human PROKR2(1-53)-hFc (SEQ IDNO:180) over the captured antibody surface. Antibody-antigen associationwas monitored for 90 seconds while dissociation was monitored for 360seconds. Kinetic on-rate (k_(a)) and off-rate (k_(d)) constants weredetermined from the data using Scrubber software version 2.0 c. Bindingdissociation equilibrium constants (K_(D)) and dissociative half-lives(t_(½)) were calculated from the kinetic rate constants as:K_(D)=k_(d)/k_(a) and t_(½)=ln(2)/k_(d). Results are shown in Tables 3(PROKR1 binding) and 4 (PROKR2 binding) (NB=no binding observed).

TABLE 3 Binding Kinetics of Anti-PROKR Antibodies binding to HumanPROKR1 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) t½ (min) H1M6386N2.12 × 10⁵ 1.03 × 10⁻³ 4.85 × 10⁻⁹ 11 H4H6385N 2.94 × 10⁵ 1.36 × 10⁻⁴4.61 × 10⁻¹⁰ 85 H4H6663P 5.83 × 10⁵ 6.20 × 10⁻³ 1.06 × 10⁻⁸ 2 H4H6669P2.36 × 10⁵ 6.23 × 10⁻⁵ 2.64 × 10⁻¹⁰ 186 H4H6671P 1.98 × 10⁵ 3.62 × 10⁻⁴1.83 × 10⁻⁹ 32 H4H6680P 2.31 × 10⁵ 4.55 × 10⁻⁵ 1.97 × 10⁻¹⁰ 254 H4H6690P5.45 × 10⁵ 1.59 × 10⁻³ 2.92 × 10⁻⁹ 7 H4H6696P 3.17 × 10⁵ 7.27 × 10⁻⁴2.30 × 10⁻⁹ 16 H4H6698P 2.55 × 10⁵ 2.87 × 10⁻⁴ 1.12 × 10⁻⁹ 40 H4H6701P3.85 × 10⁵ 4.12 × 10⁻⁴ 1.07 × 10⁻⁹ 28 H4H6706P 1.96 × 10⁵ 2.13 × 10⁻⁴1.09 × 10⁻⁹ 54

TABLE 4 Binding Kinetics of Anti-PROKR Antibodies binding to HumanPROKR2 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) t½ (min) H1M6386N NBNB NB NB H4H6385N 4.89 × 10⁵ 5.39 × 10⁻³ 1.10 × 10⁻⁸ 2 H4H6663P 1.04 ×10⁵ 1.34 × 10⁻² 1.29 × 10⁻⁷ 1 H4H6669P NB NB NB NB H4H6671P NB NB NB NBH4H6680P 5.99 × 10⁴ 2.53 × 10⁻⁴ 4.23 × 10⁻⁹ 46  H4H6690P NB NB NB NBH4H6696P 7.63 × 10⁵ 1.63 × 10⁻² 2.14 × 10⁻⁸ 1 H4H6698P 5.33 × 10⁵ 8.57 ×10⁻³ 1.61 × 10⁻⁸ 1 H4H6701P 8.91 × 10⁵ 2.15 × 10⁻³ 2.41 × 10⁻⁹ 5H4H6706P NB NB NB NB

As shown in Table 3, eleven anti-PROKR antibodies demonstrated bindingto the human PROKR1(1-62)-hFc protein with K_(D) values ranging from 197pM to 10.6 nM. As shown in Table 4, six of the 11 anti-PROKR antibodiesdemonstrated binding to the human PROKR2(1-53)-hFc protein with K_(D)values ranging from 2.41 nM to 129 nM. Five antibodies (H1M6386N,H4H6669P, H4H6671P, H4H6690P, and H4H6706P) did not demonstrate anymeasurable binding to the human PROKR2(1-53)-hFc protein in this assayformat.

Example 5 Ability of Anti-PROKR Antibodies to InhibitProkineticin-Mediated Calcium Mobilization in Cells Engineered to StablyExpress PROKR1 or PROKR2

The ability of anti-PROKR antibodies to block activation of PROKR1 andPROKR2 by their ligands prokineticin 1 (PK1) and prokineticin 2 (PK2) invitro was determined using a cell-based assay as described below.

HEK293 cells were modified to stably express either human PROKR1(293/hPROKR1), human PROKR2 (293/hPROKR2), mouse PROKR1 (293/mPROKR1),rat PROKR1 (293/rPROKR1), cynomolgus monkey PROKR1 (293/mfPROKR1) orcynomolgus monkey PROKR2 (293/mfPROKR2). For these experiments, thePROKR-expressing cell lines were maintained in complete growth medium[DME High Glucose (Irvine Scientific, #9033), 10% fetal bovine serum(Irvine Scientific, #3000A), 1% pencillin/streptomycin/glutamine (GIBCO,#10378), and 500 μg/ml G418 (GIBCO, #11811-098)].

Intracellular calcium levels were measured using a Fluo-4 NW CalciumAssay Kit (Invitrogen, #F36206). To assess the ability of anti-PROKRantibodies to inhibit human PK1-or human PK2-dependent calciummobilization, cells expressing PROKR1 or PROKR2 were seeded in 96 wellassay plates at 20,000-50,000 cells per well in complete growth mediumand allowed to grow overnight at 37° C. in 5% CO₂. The next day the cellculture medium was replaced with Fluo-4 NW kit assay buffer plus calciumindicator dye as per manufacturer's specifications. For the inhibitioncurves, anti-human PROKR antibodies were added to the cells at finalconcentrations ranging from 17 pM to 1 μm and incubated for 1 hour (30minute incubation at37° C. followed by 30 minute incubation at roomtemperature). Human PK1 (Cell Sciences; #CRV015B) or hPK2 (ProSci Inc.;#40-190) were then added for each antibody dose response to achieve aconstant ligand concentration (as shown in the corresponding tables).Relative fluorescence units (RFU) were measured every second for atleast 50 seconds using FLIPR Tetra high throughput cellular screeningsystem (Molecular Devices). For hPK1 and hPK2 dose-response curves, eachligand was added to cells without antibody at concentrations rangingfrom 70 pM to 500 nM, and then the RFU values were measured. The max—minRFU was calculated for each well and EC₅₀/IC₅₀ values were determinedfrom a four-parameter logistic equation over an 8 or 12-point responsecurve (GraphPad Prism). Results are shown in Tables 5-8. For theexperiments depicted in Table 8, 1 nM of hPK1 was the constantconcentration used in both assays.

TABLE 5 EC₅₀ Values for hPK1 or hPK2 Stimulation of HEK293 CellsExpressing Human and Monkey PROKR1 and PROKR2 And Constant LigandConcentrations Used for IC₅₀ Determinations EC₅₀ (nM)/Ligand Constant(nM) Human Human Monkey Monkey PROKR1 PROKR2 PROKR1 PROKR2 hPK1Experiment 1  6.2/20 ND 69/20 ND ligand Experiment 2 14.3/20  8.7/10 NDND Experiment 3 2.2/1 12.7/20 5/5 22.6/25 hPK2 Experiment 1 30.7/20 NDND ND ligand Experiment 2 ND 156/9  ND ND Experiment 3 0.6/1 3.7/50.1/0.5  0.3/0.5

TABLE 6 IC₅₀ Values for Anti-PROKR Antibody Inhibition of Calcium Fluxin hPK1- or hPK2-Stimulated HEK293 Cells Expressing Human PROKR1 andHuman PROKR2 hPROKR1 + PK1 hPROKR1 + PK2 hPROKR2 + PK1 hPROKR2 + PK2IC₅₀ Block IC₅₀ Block IC₅₀ Block IC₅₀ Block Antibody (nM) (%) (nM) (%)(nM) (%) (nM) (%) H1M6386N 14.9*   92.03*   16.2* 66.60*  ND  ND  ND  NDH4H6663P >1000**     15.48** ND ND NB** 0** NB** 0**  H4H6669P 14**   52.42** ND ND NB** 0** NB** 0**  H4H6671P 14.5**  45.00** ND ND NB** 0**NB** 0**  H4H6680P 16.4**  51.05** ND ND NB** 0** NB** 0** H4H6690P >1000**     24.70** ND ND NB** 0** NB** 0**  H4H6696P 13.1*** 101.04*** >300*** 32.85***  NB***  0***  NB*** 0*** H4H6698P 8.5***100.58*** >300*** 40.03***  NB***  0***  NB*** 0*** H4H6701P 6.6*** 98.55*** >300*** 56.62*** >300***   48.56*** >300*** 60.80***  H4H6706P 11.2**  59.12** ND ND NB** 0** NB** 0**  H4H6385N 6.3***100.89***   55.1*** 77.52***  NB***  0***  NB*** 0*** ND: not determinedNB: no blocking observed *= Used constant ligand concentration shown forExperiment 1 in Table 5 **= Used constant ligand concentration shown forExperiment 2 in Table 5 ***= Used constant ligand concentration shownfor Experiment 3 in Table 5

TABLE 7 IC₅₀ Values for Anti-PROKR Antibody Inhibition of Calcium Fluxin hPK1- or hPK2-Stimulated HEK293 Cells Expressing Monkey PROKR1 andMonkey PROKR2 MfPROKR1 + PK1 MfPROKR1 + PK2 MfPROKR2 + PK1 MfPROKR2 +PK2 IC₅₀ Block IC₅₀ Block IC₅₀ Block IC₅₀ Block Antibody (nM) (%) (nM)(%) (nM) (%) (nM) (%) H1M6386N NB* 0*   ND ND ND   ND ND   ND H4H6696P31***    93.91*** >300*** 67.92*** NB*** 0*** NB*** 0*** H4H6698P32.9***   99.34***    63.9*** 85.74*** NB*** 0*** NB*** 0*** H4H6701P6.4*** 100.67*** >300*** 50.53*** >300***   21.36*** >300***   61.21***H4H6385N 6.7*** 100.82***    51.8*** 93.70*** NB*** 0*** NB*** 0*** ND:not determined NB: no blocking observed *= Used constant ligandconcentration shown for Experiment 1 in Table 5 ***= Used constantligand concentration shown for Experiment 3 in Table 5

TABLE 8 IC₅₀ Values for Anti-PROKR Antibody Inhibition of Calcium Fluxin hPK1-Stimulated HEK293 Cells Expressing Mouse PROKR1 and Rat PROKR1mPROKR1 + PK1 rPROKR1 + PK1 Antibody IC₅₀ (nM)* Block (%) IC₅₀ (nM)**Block (%) H4H6669P NB 0 ND ND H4H6671P NB 0 ND ND H4H6680P NB 0 ND NDH4H6696P NB 0 NB 0 H4H6698P NB 0 NB 0 H4H6701P >300 20.85 NB 0 H4H6385NNB 0 NB 0 ND: not determined NB: no blocking observed *= Observed hPK1EC₅₀ value (no antibody) was 0.9 nM **= Observed hPK1 EC₅₀ value (noantibody) was 0.7 nM

Eleven anti-PROKR1 antibodies were tested for inhibition ofhPK1-mediated calcium mobilization in 293/hPROKR1 cells. As shown Table6, four antibodies (H4HM6385N, H4H6701P, H4H6698P, and H4H6696P)blocked >98% of the PROKR1 activity with IC₅₀ values ranging from 6.3 nMto 13.1 nM. Another anti-PROKR antibody, H1M6386N, blocked >92% of thehPK1 mediated calcium mobilization of 293/hPROKR1 cells with an IC₅₀value of 14.9 nM. Four anti-PROKR antibodies (H4H6669P, H4H6671P,H4H6680P, H4H6706P) blocked between 45% to 60% of the hPK1-mediatedcalcium mobilization in 293/hPROKR1 cells with IC₅₀ values ranging from11.2 nM to 16.4 nM. Two anti-PROKR antibodies (H4H6663P and H4H6690P)blocked less than 25% of the hPK1-mediated calcium mobilization in293/hPROKR1 cells.

Five anti-PROKR antibodies were also tested for inhibition ofhPK2-mediated calcium mobilization in 293/hPROKR1 cells as shown inTable 6. One anti-PROKR antibody, H4HM6385N, blocked >77% of thehPK2-mediated calcium mobilization in 293/hPROKR1 cells with an IC₅₀value of 55.1 nM, while 2 anti-PROKR antibodies (H1M6386N and H 4H6701P)blocked approximately 67% to 57% of the calcium mobilization with IC₅₀values of 16.2 nM and >300 nM. Two anti-PROKR antibodies (H4H6696P andH4H6698P) blocked approximately 40% and 33% of the hPK2-mediated calciummobilization in 293/hPROKR1 cells with IC₅₀ values>300 nM.

Ten anti-PROKR antibodies were also tested for inhibition of humanPROKR2 function, as shown in Table 6. H4H6701P blocked hPK1-orhPK2-mediated calcium mobilization of 293/hPROKR2 cells under theseassay conditions. This antibody blocked approximately 49% of the hPK1-mediated calcium mobilization and approximately 61% of thehPK2-mediated calcium mobilization with IC₅₀ values>300 nM for both.None of the other tested antibodies blocked hPK1-or hPK2-mediatedcalcium mobilization of 293/hPROKR2 cells.

Five of the anti-PROKR antibodies were further tested for their abilityto inhibit hPK1-mediated calcium flux in cells expressing cynomolgusmonkey PROKR1 as shown in Table 7. Four antibodies (H4H6385N, H4H6701P,H4H6696P, and H4H6698P) blocked >90% of the hPK1-mediated calciummobilization in 293/MfPROKR1 cells with IC₅₀ values ranging from 6.4 nMto 32.9 nM. One antibody, H1M6386N, did not measurably blockhPK1-mediated calcium mobilization in 293/MfPROKR 1 cells. Four of theanti-PROKR antibodies were also tested for their ability to inhibithPK2-mediated calcium flux in cells expressing cynomolgus monkey PROKR1as shown in Table 7. One anti-PROKR antibody, H4H6385N blockedapproximately 94% of hPK2-mediated calcium mobilization in 293/MfPROKR1cells with an IC₅₀ value of 51.8 nM. Another anti-PROKR antibody,H4H6698P, blocked approximated 86% of the hPK2-mediated calciummobilization in 293/MfPROKR1 cells with an IC₅₀ value of 63.9 nM. Thetwo other anti-PROKR antibodies tested (H4H6696P and H4H6701P) blockedapproximately 68% and 51% of the hPK2-mediated calcium mobilization in293/MfPROKR1 cells with an IC₅₀ values>300 nM. These four antibodieswere also tested for their ability to inhibit hPK1-or hPK2-mediatedcalcium flux in cells expressing monkey PROKR2 as shown in Table 7.H4H6701P blocked approximately 21% of the hPK1-mediated calciummobilization and blocked 61% of the hPK2-mediated calcium mobilizationwith IC₅₀ values>300 nM for both. The other three tested antibodies didnot block hPK1-or hPK2-mediated calcium mobilization of 293/MfPROKR2cells under these assay conditions.

Seven of the anti-PROKR antibodies were tested for their ability toblock hPK1-mediated calcium flux in cells expressing mouse PROKR1 asshown in Table 8. H4H6701P blocked approximately 21% of thehPK1-mediated calcium mobilization of 293/mPROKR1 cells with an IC₅₀value>300 nM, and the other 6 tested antibodies did not block in thisassay. Four of the anti-PROKR antibodies were tested for their abilityto block hPK1-mediated calcium flux in cells expressing rat PROKR1 asshown in Table 8. None of the tested anti-PROKR antibodies blockedstimulation of 293/rPROKR1 by hPK1 under these assay conditions.

Example 6 Ability of Anti-PROKR Antibodies to InhibitProkineticin-Mediated Calcium Mobilization in Cells Engineered ForInducible Expression of PROKR1 or PROKR2

The ability of anti-human PROKR antibodies to block activation ofprokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2) bytheir ligands prokineticin 1 (PK1) and prokineticin 2 (PK2) in vitro wasdetermined using a cell-based assay as described below.

CHO cells were modified for inducible expression of either human PROKR1(CHO/hPROKR1), human PROKR2 (CHO/hPROKR2), mouse PROKR1 (CHO/mPROKR1),or mouse PROKR2 (CHO/mPROKR2). For these experiments, thePROKR-expressing cell lines were generated and maintained in completegrowth medium [DME High Glucose (Irvine Scientific, #9033), 10% fetalbovine serum (Irvine Scientific, #3000A), 1%penicillin/streptomycin/glutamine (GIBCO, #10378), and 500 μg/mL G418(GIBCO, #11811-098)]. To induce PROKR expression, CHO cell lines weregrown in the presence of 0.5 mg/mL doxycycline for 16 to 24 hours.Non-induced cells were handled in an identical manner, but in theabsence of doxycycline. As determined by FACS, PROKR surface stainingwas present on CHO cells even in the absence of the inducer, but at alower level than in the presence of the inducer.

Intracellular calcium levels were measured using a Fluo-4 NW CalciumAssay Kit (Invitrogen, #F36206) as per the manufacturer'sspecifications. To assess the ability of anti-PROKR antibodies toinhibit human PK1-or human PK2-dependent calcium mobilization, CHO celllines were grown in the presence or absence of doxycycline. Cells werethen seeded in 96 well assay plates at 125,000 cells per well in Fluo-4NM assay buffer, incubated for 1 hour at 37° C. in 5% CO₂, and anequivalent volume of Fluo-4 NW kit assay buffer plus calcium indicatordye was then added to each well. For the inhibition curves, anti-humanPROKR antibodies were added to the cells at final concentrations rangingfrom 1.0 μM to 1.3 nM and incubated for 1 hour (30 minute incubation at37° C. followed by 30 minute incubation at room temperature). Constantconcentrations of hPK1 or hPK2 (as shown in the corresponding figures)were then added to cells that had been pre-incubated with antibody, andrelative fluorescence units (RFU) were measured every second for atleast 50 seconds using FLIPR Tetra (Molecular Devices). For hPK1 andhPK2 dose-response curves, each ligand was added to cells withoutantibody at concentrations ranging from 10 pM to 300 nM, and RFU weremeasured as for the antibody inhibition curves. The max—min RFU wascalculated for each concentration and EC₅₀/IC₅₀ values were determinedfrom a four-parameter logistic equation over an 8 or 12-point responsecurve (GraphPad Prism). Ligand EC₅₀ values or mean EC₅₀ values (±SEM)are shown in Table 9.

TABLE 9 Ligand EC₅₀ Values on Non-induced and Induced CHO/hPROKR1,CHO/hPROKR2, CHO/mPROKR1, and CHO/mPROKR2 Cell Lines Ligand EC₅₀ (nM)Cell line hPK1 hPK2 CHO/hPROKR1 (non-induced) 48 (±14.3)  1.4 (±0.58)CHO/hPROKR1 (induced) 2.0 (±0.82)   1.6 (±0.96) CHO/hPROKR2 (induced) 40(±34.95) 9.8 (±9.4)  CHO/mPROKR1 (non-induced) 8.3 0.8 CHO/mPROKR1(induced) 1.5 9.6 CHO/mPROKR2 (non-induced) 112 18 CHO/mPROKR2 (induced)4.5 2.2

Four anti-PROKR 1 antibodies were tested for inhibition of hPK1 andhPK2-mediated calcium mobilization in non-induced or inducedCHO/hPROKR1, CHO/hPROKR2, CHO/mPROKR1, and CHO/mPROKR2 cell lines.Blocking results in the non-induced CHO/hPROKR1 cell line are shown inTable 10.

TABLE 10 Blocking of hPK1 and hPK2 Induced Calcium Flux on Non-InducedCHO/hPROKR1 Cell Line CHO/hPROKR1 non-induced IC₅₀ (nM) Antibody PK1 PK2H4H6385N 14.2 (±9.43)* 20.0 (±10.4)*** H4H6701P 4.1** 21.9*** H4H6696P3.8** 19.1*** H4H6698P 3.1** 27.4*** *IC₅₀ values for H4H6385N are anaverage of experiments that used either 40 nM or 80 nM constantconcentration of hPK1 **Experiment used 40 nM constant concentration ofhPK1 ***Experiment used 4 nM constant concentration of hPK2

As shown in Table 10, all four antibodies tested (H4HM6385N, H4H6701P,H4H6696P, and H4H6698P) blocked hPK1 mediated calcium flux in thenon-induced CHO/hPROKR1 cell line (low hPROKR1 expressing cells) to nearbaseline levels with IC₅₀ values ranging from 3.1 nM to 14.2 nM. Thesesame antibodies also blocked hPK2-mediated calcium flux in thenon-induced CHO/hPROKR1 cell line to near baseline line levels with IC₅₀values ranging from 19.1 nM to 27.4 nM. No blocking was observed in theinduced CHO/hPROKR1 cell line (high expressing hPROKR1 cells) followingligand activation with either PK1 or PK2. None of the antibodies testeddemonstrated blockade of hPK1 or hPK2-mediated calcium flux in theinduced CHO/hPROKR2 cell line. Ligand activation of non-inducedCHO/hPROKR2 cells was weak and inconsistent (data not shown), soantibody blockade could not be evaluated. None of the anti-PROKRantibodies tested blocked stimulation of non-induced or inducedCHO/mPROKR1 and CHO/mPROKR2 cell lines with hPK1 or hPK2 under identicalassay conditions.

The results of this Example confirm that anti-PROKR antibodies H4H6385N,H4H6701P, H4H6696P and H4H6698P effectively block PK1-and PK2-mediatedPROKR1 signaling.

Example 7 Efficacy of an Anti-PROKR Antibody in a Model of DSS-InducedColitis

In this Example, the ability of the anti-PROKR antibody H4H6385N toattenuate changes in open field behaviors following 7 days of oraldextran sodium sulfate administration (DSS, 4% w/v in drinking water) orwater control was assessed.

Humanized Prokr1 mice, in which the coding sequence of the mouse Prokr1gene was replaced with the corresponding human PROKR1 sequence, wereused in this experiment (mixed male and female, 21-31 weeks of age).Separate cohorts of mice received 30 mg/kg (s.c.) of an isotype controlantibody, 30 mg/kg (s.c.) of H4H6385N, or no injection. DSSadministration was initiated 24 hours after antibody dosing. All micewere then tested in an automated open field apparatus (Kinder ScientificSmartFrame, Poway, Calif.).

It has been previously observed that DSS-induced colitis reliably alters4 parameters of the open field assay: time spent immobile (immobility),the total amount of exploratory activity (total distance), the number oftimes that the mouse rears onto its hind limbs (rearing) and the amountof time it spends rearing onto its hind limbs (rearing time). Theresults of this experiment, expressed as the sum of the total time spentperforming each behavior or the total counts of each behavior (asappropriate) of over the test period of 60 minutes, are shown in FIGS.1A-1D (all data are represented as group mean±SEM. Each cohort isn=8-9/group).

As summarized in FIGS. 1A-1D, mice treated with the exemplary anti-PROKRantibody H4H6385N prior to DSS administration exhibited improved openfield behaviors (i.e., reduced immobility, increased total distance, andincreased rearing) as compared to untreated and isotype control-treatedmice subjected to equivalent DSS administration conditions.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that specifically binds cell surface-expressed prokineticinreceptor 1 (PROKR1) and cell surface-expressed prokineticin receptor 2(PROKR2) and blocks prokineticin-mediated activation of the PROKR1,wherein the antibody or antigen-binding fragment comprises three heavychain complementarity determining regions (CDRs) of a heavy chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 18; and three light chain CDRs of a light chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO:26.
 2. Theantibody or antigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment thereof blocks prokineticin-1 (PK1)-mediatedactivation of PROKR1.
 3. The antibody or antigen-binding fragment ofclaim 1, wherein the antibody or antigen-binding fragment thereof blocksprokineticin-2 (PK2)-mediated activation of PROKR1.
 4. The antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment thereof blocks prokineticin-1 (PK1)-mediatedactivation of PROKR1 and prokineticin-2 (PK2)-mediated activation ofPROKR1.
 5. The antibody or antigen-binding fragment of claim 2, whereinthe antibody or antigen-binding fragment thereof blocks PK1-mediatedactivation of PROKR1 with an IC₅₀ of less than 20 nM, as measured in acalcium mobilization assay using PROKR1-expressing cells stimulated with1 to 20 nM PK1 in vitro.
 6. The antibody or antigen-binding fragment ofclaim 5, wherein the antibody or antigen-binding fragment thereof blocksPK1-mediated activation of PROKR1 with an IC₅₀ of less than 10 nM, asmeasured in a calcium mobilization assay using PROKR1-expressing cellsstimulated with 1 to 20 nM PK1 in vitro.
 7. The antibody orantigen-binding fragment of claim 3, wherein the antibody orantigen-binding fragment thereof blocks PK2-mediated activation ofPROKR1 with an IC₅₀ of less than 60 nM, as measured in a calciummobilization assay using PROKR1-expressing cells stimulated with 1 to 20nM PK2 in vitro.
 8. The antibody or antigen-binding fragment of claim 7,wherein the antibody or antigen-binding fragment thereof blocksPK2-mediated activation of PROKR1 with an IC₅₀of less than 20 nM, asmeasured in a calcium mobilization assay using PROKR1-expressing cellsstimulated with 1 to 20 nM PK2 in vitro.
 9. The isolated antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment comprises: HCDR1 comprising the amino acidsequence set forth in SEQ ID NO: 20; HCDR2 comprising the amino acidsequence set forth in SEQ ID NO: 22; HCDR3 comprising the amino acidsequence set forth in SEQ ID NO: 24; LCDR1 comprising the amino acidsequence set forth in SEQ ID NO: 28; LCDR2 comprising the amino acidsequence set forth in SEQ ID NO: 30; and LCDR3 comprising the amino acidsequence set forth in SEQ ID NO:
 32. 10. The antibody or antigen-bindingfragment of claim 9, wherein the antibody or antigen-binding fragmentcomprises: (a) a heavy chain variable region (HCVR) comprising the aminoacid sequence set forth in SEQ ID NO: 18; and (b) a light chain variableregion (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:26.
 11. A pharmaceutical composition comprising the antibody orantigen-binding fragment of claim 1, and a pharmaceutically acceptablecarrier or diluent.
 12. A composition comprising the antibody orantigen-binding fragment of claim 1 and one or more additionaltherapeutic agents.
 13. The composition of claim 12, wherein theadditional therapeutic agent is selected from the group consisting of anantiviral, an antibiotic, an analgesic, a corticosteroid, an opioid, andan NSAID (non-steroidal anti-inflammatory drug).
 14. The composition ofclaim 12, wherein the additional therapeutic agent is selected from thegroup consisting of: an anti-NGF antibody, an anti-PAR2antibody, ananti-ASIC1 antibody, an anti-ASIC2 antibody, an anti-ASIC3 antibody, ananti-ASIC4 antibody, an anti-GFR alpha antibody, an anti-EGFR antibody,an anti-ErbB2 antibody, anti-ErbB3 antibody, an anti-ErbB4 antibody, ananti-EGFRvIII antibody, an anti-cMET antibody, an anti-IGF1R antibody,an anti-PDGFR-alpha antibody, an anti-PDGFR-beta antibody, a VEGF-Trap,an anti-VEGF antibody, an anti-DLL4 antibody, an anti-Ang2 antibody, ananti-FOLH1 antibody, an anti-PRLR antibody, an anti-STEAP1 antibody, ananti-STEAP2 antibody, an anti-TMPRSS2 antibody, an anti-MSLN antibody,an anti-CA9 antibody, an anti-uroplakin antibody, an anti-CD20 antibody,an anti-IL-1 antibody, an anti-IL-2 antibody, an anti-IL-3 antibody, ananti-IL-4 antibody, an anti-IL-5 antibody, an anti-IL-6 antibody, ananti-IL-8 antibody, an anti-IL-9 antibody, an anti-IL-11 antibody, ananti-IL-12 antibody, an anti-IL-13 antibody, an anti-IL-17 antibody, ananti-IL-18 antibody, an anti-IgE antibody, and an anti-TNF antibody.